Overexpression of TNF alpha (tumor necrosis factor alpha) in the human body is known to cause autoimmune diseases. TNFR (tumor necrosis factor receptor) is a TNF alpha receptor, and binds to an overexpressed TNF alpha to function as a therapeutic agent for autoimmune diseases. In addition, TNFR is fused with the human immunoglobulin G (IgG) Fc region to be expressed as an Fc fusion protein, thereby being used as a therapeutic protein drug.
TNFR-Fc can be prepared by fusion of 235 amino acids of TNFR with 232 amino acids of the Fc region including a hinge region. When TNFR-Fc is produced as a dimer by recombinant DNA technology, it shows a biological activity.
TNFR comprises 235 amino acids possessing 4 domains and a transmembrane region. TNFR has 22 cysteines, and all of them form disulfide bonds to have a steric structure. However, when TNFR-Fc is produced from animal cells, cysteines bind with each other at random, and thus they do not form disulfide bonds identical to those of a native protein. TNFR may be also partially truncated, and fail to form a correct TNFR-Fc dimer.
TNFR-Fc with incorrect disulfide bonds cannot show the proper biological activity due to a drastic reduction in the binding ability to TNF alpha. When the entire or a part of TNFR is truncated, it may also not exhibit the biological activity.
Therefore, when the TNFR-Fc dimers are produced using a recombinant DNA technology and an animal cell culture technique, active proteins, inactive proteins with incorrect disulfide bonds, aggregates, and clipped forms are produced at the same time, and thus a technique for isolating active proteins from the protein mixture is needed.
Therapeutic biomolecules must be purified with greater than 99% purity prior to human use. This degree of purification can be achieved through use of three or four liquid chromatography processes such as ion exchange chromatography, reversed phase chromatography, size exclusion chromatography, affinity (dye, metal, antibody, protein A, etc.) chromatography, and hydrophobic interaction chromatography which are required during the isolation process. The type and sequence of chromatographic processes chosen are based on physicochemical characteristics of contaminants that coexist with the target biomolecule. Usually, size exclusion chromatography is employed in a final step, because it removes protein aggregates and also exchanges the purified proteins into the final formulation buffer.
However, size exclusion chromatography negatively affects productivity because of the limited sample volume that can be loaded. Column load volume of sample greater than about 5% of the overall column volume results in diffusion-related band spreading and dilution as the solute band moves through the column. The volume limitation can be circumvented through the use of an ultrafiltration concentration step prior to column loading. Ultrafiltration concentration also introduces productivity losses due to non-specific binding of the protein to the membrane and other materials in the system, volume losses to tubing and pumps, and complications associated with equipment preparation, operation, and cleaning. Therefore, the purification process can clearly be simpler and productivity losses can be avoided by applying a concentrated target protein to hydrophobic interaction chromatography to obtain displacement effect. However, the separation method of the target protein using hydrophobic interaction chromatography requires different separation conditions depending on the type of the target protein, the expression vector including a polynucleotide encoding the protein, etc. Frequently, the known separation methods of the target protein cannot be applied in the separation of other target proteins or in the separation of the target protein prepared under different conditions. In the market for protein drugs, therefore, it is very important to investigate suitable conditions for separating a desired target protein with high purity and concentration.